Aps coated glass slides

Field-collected samples were washed with tap water to remove soil. Safranin-O staining of whole haustoria was performed as previously described (Yoshida and Shirasu 2009 (link)). For histological sections, haustoria and infected host roots were separated and fixed with FAA solution (10% formaldehyde, 5% acetic acid, 50% ethanol) under vacuum for 15 min followed by incubation at room temperature for 2 h. Samples were subjected to an ethanol dehydration series and embedded into Technovit 7100 resin (Heraeus Kulzer GmbH) according to the manufacturer’s instruction. Solidified resin blocks were mounted on wood blocks with Technovit 3040 (Heraeus Kulzer GmbH), then sectioned using a microtome (Leica, RM2135) to 4 μm thickness. Sections were stained with 0.1% Safranin-O (Sigma-Aldrich) and counterstained with 0.1% Fast Green (Wako Chemical) on APS-coated glass slides (Matsunami Glass).

PBMCs from ATL patients were washed and centrifuged onto APS-coated glass slides (Matsunami Glass) by Cytospin (Shandon). Cells were then fixed with 4% paraformaldehyde, permeabilized, and blocked with Duolink Blocking Solution. After that, cells were stained with mouse anti-HBZ antibody (1A10, generated by immunizing C57BL/6 mice with keyhole limpet hemocyanin [KLH]- conjugated HBZ peptide 97-133 [CKQIAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQE]) or mouse anti-Smad3 (7F3, Sigma-Aldrich) and rabbit anti-human APOBEC3G antibody (D9C6Z, Cell Signaling Technology), which were diluted in Duolink Antibody Diluent, followed by incubation with Duolink In Situ PLA Probe Anti-Mouse PLUS and Duolink In Situ PLA Probe Anti-Rabbit MINUS. Next, cells were incubated with Duolink 5× Ligation Buffer and Duolink Polymerase (10 u/µL), followed by incubation with Duolink Amplification Red and Duolink Ligase (1 U µL). The stained cells were mounted with Duolink®In Situ Mounting Medium with DAPI. The cells were imaged using a C2 confocal microscope (Nikon) and analyzed with ImageJ.

Frozen sections were cut at a 10-μm thickness and mounted on either poly-L-lysine-coated or APS-coated glass slides (Matsunami, Osaka, Japan). The sections were then fixed with ethanol at −20°C for 30 min and washed with phosphate-buffered saline (PBS). The sections were next incubated with 5% normal donkey serum-PBS to block non-specific antibody binding, followed by sequential incubations in primary and secondary antibodies diluted in 5% normal donkey serum-PBS. For nuclear staining, 4'6-damidino-2-phenylindole (DAPI) was added to the secondary antibody solution. The final stained specimens were mounted with Vectashield (Cat. #H-1000; Vector Laboratories, Newark, CA) or handmade mounting medium containing 1,4-Diazabicyclo[2.2.2]octane, and examined under a BX62 microscope equipped with Nomarski differential interference-contrast and epifluorescence optics (Olympus, Tokyo, Japan). Images were captured using a CoolSNAP K4 CCD camera (Photometrics, Tucson, AZ) and MetaMorph software version 6.1 (Molecular Devices, Silicon Valley, CA).
In histochemical controls, the sections were processed in the same way as those incubated with each antibody, except omitting primary antibody from primary antibody solutions; and we confirmed that the labeling detected by each antibody was not observed in these negative control specimens (data not shown).

Sense or antisense NIPSNAP1 riboprobe RNAs were synthesized in vitro using digoxigenin-11-UTP (Roche) with a 700-bp coding region of NIPSNAP1 as template. A sliding microtome was used to prepare spinal cord sections (40 µm) from paraformaldehyde-perfused mice. DRG sections (10 µm) prepared using a cryostat were mounted on APS-coated glass slides (Matsunami). The free-floating spinal cord and DRG tissue sections on glass slides were hybridized overnight with digoxigenin-labeled riboprobes at 65℃ in a solution containing 50% formamide, 4% dextran sulfate, 250 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA, 1× Denhardt’s solution, 0.2% SDS, 25 mM EDTA, 750 mM NaCl, and 25 mM PIPES. Hybridized sections were then washed with 0.1× SCC after treatment with 20 µg/ml RNase A for 30 min at 37℃. The sections were then reacted with an alkaline phosphate-conjugated anti-digoxigenin antibody, and hybrids were detected using 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate (Roche), 4-chloro-2-metylbenzenediazonium hemi-zinc chloride salt (Fast Red TR, Roche), and levamisole (Sigma-Aldrich). Thereafter, the free-floating spinal cord was mounted on glass slides. Digital images were captured using an LSM510 Zeiss laser-scanning confocal microscope equipped with an argon He–Ne laser and the appropriate filter.